1. Field of Invention
This invention relates to polyurethanes modified to retain epithelial cells. Particularly, it relates to implants made from such polyurethanes.
2. Description of Related Art
Polyurethanes, i.e. polymers which comprise repeating units having a urethane group in the polymer backbone, can be used to form bulk polymers, coatings, fillings, and films. Notably, polyurethanes are also readily machinable once set. These properties of polyurethanes have rendered them useful for medical and non-medical purposes.
With regard to medical applications, polyurethanes are widely used in implants, particularly cardiovascular implants, as highly biocompatible biomaterials. For example, polyurethanes have been employed in the manufacture of pacemaker electrodes, vascular grafts, and artificial heart valves.
Medical uses of polyurethanes have, however, been heretofore limited by, among other reasons, the tendency of polyurethane products which contact the blood stream or other biological fluids to calcify, induce thrombogenesis, and/or impede cellular attachment and growth. Promotion of cell adhesion on medical devices comprising polyurethane is of particular concern, as the establishment of a confluent monolayer of endothelial cells is critical for neovascularization (Simionescu, N. and N. Simionescu, eds. Endothelial Cell Dysfunctions. 1992. New York:Plenum Press). Additionally, the cellular attachment to vascular grafts or implants must be strong enough to withstand the fluid shear stress exerted by blood and other biological fluids. Modified polyurethane surfaces currently available, such as those that have been deliberately textured or cast as foams, have provided for only disorganized cell growth. Polyurethane surfaces have also been coated with fibronectin and collagen which are potent ligands for cell surface receptors. Despite promoting cell adhesion, fibronectin and collagen coated polyurethanes are not ideal modifications because apoptosis and calcification of vascular cells has been observed when cultivated on collagen coated matrices and vascular cells seeded on fibronectin coated polyurethane failed to thrive.
As those skilled in the art will appreciate, a need exists for implantable devices comprising polyurethane which allows for enhanced cellular attachment and growth.
Natural blood contact surfaces, such as those found within blood vessels, possess mechanisms that prevent blood from clotting during normal passage along the surface. In the case of a mammalian artery, the immediate blood contact surface comprises a layer of non-thrombogenic ECs. Immediately external to the EC layer is the remainder of the intima: a subendothelial matrix layer of basement membrane and underlying glycoprotein-bearing extracellular matrix, and the internal elastic lamina. Surrounding the intima layer is the multilaminate media structure containing smooth muscle cells (SMCs) and elastin, and surrounding this media is the adventitia, the most external layer comprised of fibroblasts and connective tissue. Both the subendothelial layer and media are generally considered to be thrombogenic in nature in order to maintain hemostasis when the vascular system is injured.
Intact endothelial linings are considered to be non-thrombogenic unless damaged. Because of their blood-contacting location, ECs have been thoroughly investigated with respect to their anti-thrombogenic function. ECs are known to synthesize or bind a number of substances with coagulation-inhibiting or fibrinolytic function including heparan sulfate/antithrombin III, dermatan sulfate/heparin cofactor II, thrombomodulin/protein C/protein S, prostacyclin and tissue-type plasminogen activator. Furthermore, segments of endothelium-bearing autologous vessel transplanted from one site to another in an individual to bypass diseased vessels exhibit an incidence of thrombosis substantially less than that of synthetics used in the same application. For these reasons, it has been assumed that ECs are responsible for the non-thrombogenic activity of vessels. According to this assumption, synthetic blood contact devices capable of thrombo-resistance similar to the native vasculature would require a blood contact surface of ECs.
Numerous attempts have been made to provide prosthetic surfaces, and specifically vascular grafts, that include or develop an EC lining as described in U.S. Pat. No. 5,879,383 to Bruchman et al. The overwhelming majority of these attempts have been carried out as an intraoperative cell-seeding procedure. Intraoperative cell-seeding typically involves harvesting ECs from the recipient during the procedure and immediately seeding these collected cells onto a vascular graft that has been pretreated with a substrate to enhance EC attachment. Substrates frequently applied to the synthetic surface include preclotted blood taken from the patient or extracellular matrix proteins such as fibronecfin, collagen, or laminin, either singly or in combination. This approach was first reported by M. B. Herring et al. in “A single staged technique for seeding vascular grafts with autogenous endothelium,” Surgery 84:498-504 (1978). In this procedure, autologous cells were seeded onto a preclotted DACRON™ graft. These seeded grafts demonstrated a decreased thrombus formation compared to control grafts without ECs. In spite of the improved initial adherence of the cells to the synthetic surface afforded through the use of these various substrata, the shear forces resulting from blood flow nevertheless leads to the loss of a substantial fraction of the applied cells.
In a variation of the above, U.S. Pat. No. 4,960,423 to Smith describes the use of elastin-derived peptides to enhance EC attachment. Some studies have used combinations of extracellular matrix molecules and cells to provide a substrate for EC attachment and growth. For example, U.S. Pat. Nos. 4,539,716 and 4,546,500 to Bell describe means by which ECs are grown on a living smooth muscle cell-collagen lattice. In addition, U.S. Pat. No. 4,883,755 to Carabasi et al. describes a technical method for seeding ECs onto damaged blood vessel surfaces.
Alternative means of growing ECs on vascular grafts have also been reported. For example, the use of physical force to apply ECs to graft surfaces is described in U.S. Pat. No. 5,037,378 to Muller et al. In another example, U.S. Pat. No. 4,804,382 to Turina and Bittman describe the application of ECs to a semi-permeable membrane in which the pores are filled with aqueous gels to allow EC coverage.
Another approach has been the seeding of ECs onto biologically-derived surfaces, including pericardium, cardiac valve leaflets, amnion, and arteries. These efforts appear to have originated with J. Hoch et al. in “In vitro endothelialization of an aldehyde-stabilized native vessel,” J. Surg. Res. 44:545-554 (1988), where the authors attempted to grow endothelium on ficin-digested, dialdehyde stabilized, bovine artery. Human venous ECs were found to adhere to and spread on the remnant collagen surface of these enzyme-digested grafts, but no implant studies were performed. J. Hoch et al. also investigated the growth of endothelium on human amnion, on live, mechanically-scraped human artery, and again on ficin-digested, tanned bovine artery. (J. Hoch et al., “Endothelial cell interactions with native surfaces,” Ann. Vase. Surg. 2:153-159 (1989)). Although EC adhesion was observed on these surfaces by 2 hours, the longterm persistence of ECs on these surfaces was not examined, and, as in the previous study, none of these endothelialized materials were implanted as vascular substitutes. Schneider et al. showed that ECs could be successfully seeded onto the remaining collagenous surface of baboon vessels from which the intima was removed. (P. A. Schneider et al., “Confluent durable endothelialization of endarterectomized baboon aorta by early attachment of cultured endothelial cells,” J. Vasc. Surg. 11:365-372 (1990)). Lalka et al. used detergent extraction of canine arteries to produce an ethanol-fixed acellular vascular matrix onto which human umbilical vein ECs were successfully seeded in vitro. (S. G. Lalka et al., “Acellular vascular matrix: A natural endothelial cell substrate,” Ann. Vasc. Surg. 2:108-117 (1989)).
Although a small number of grafts seeded lumenally with ECs have been implanted clinically outside of the United States, and improved patencies over non-seeded grafts have been observed, this approach has generally enjoyed mixed success, and the concept still faces many challenges as described in U.S. Pat. No. 6,733,747 to Anderson et al. First, it is necessary that the cells used to seed the graft be autologous or otherwise non-immunogenic to avoid recognition and destruction of the cells by the patient's immune system. To obtain autologous ECs from a patient, the cells must be harvested from an isolated blood vessel. The harvesting surgical procedure not only increases prosthetic implant preparation time, but can also lead to complications and discomfort for the patient.
Second, retention of the cells on the graft surface after implantation has been an issue. A number of methods have been disclosed to address this issue, and include forcible injection of ECs into the graft, preclotting and seeding the lumenal surface of the graft, static adhesion-seeding of the lumen, vacuum seeding of the lumen, seeding the lumen in an extracellular matrix, and seeding of the lumen using electrostatic and gravitational forces. These methods are reviewed or disclosed in more detail in U.S. Pat. No. 5,723,324 to Bowlin et al. Additionally, it has been suggested that flow conditioning the seeded graft in vitro prior to implantation would improve cell retention by allowing the cells to secrete adhesion factors in response to slowly increasing shear rates (Dardik et al., 1999, J Vasc Surg 29: 157-67; Ballerman et al., 1995, Blood Purif 13: 125-34; and Ott and Ballerman, 1995, Surgery 117: 334-9). Although there is some evidence that methods such as conditioning may improve cell retention, all of these methods add yet another level of complexity to the seeding process and it is still not clear that significantly improved cellular retention can be achieved.
Thus, there remains a need in the art for a better EC seeding technique of prostheses in order to provide long-term patency and eventual healing by inhibiting thrombosis and inflammatory cell interactions. The instant invention provides a novel method of EC seeding by utilizing a cholesterol modified polyurethane surface to which the ECs can attach.
All references cited herein are incorporated herein by reference in their entireties.